The properties and function of NADH-semidehydroascorbate reductase in microsomal and mitochondrial fractions

Kristjansson, Elin Olafsdottir (1971) The properties and function of NADH-semidehydroascorbate reductase in microsomal and mitochondrial fractions. Masters thesis, Memorial University of Newfoundland.

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Abstract

The involvement of cytochrome b₅ reductase and cytochrome b₅ in the activity of semidehydroascorbate (AH*) reductase has been investigated by comparing the properties of AH* reductase to those of ferricyanide reductase and rotenone insensitive cytochrome c reductase. -- The AH* and cytochrome c reductases were localized in the microsomal and outer mitochondrial membrane fractions, a localization which coincides with that of cytochrome b₅ reductase. -- The AH* reductase showed a Km value for NADH of 1 μM and a pH optimum of 7.4, which agrees favourably with that of ferricyanide and cytochrome c reductases. The AH* and cytochrome c reductases showed a similar sensitivity to sulphydryl inhibition as ferricyanide reductase and all three enzjnnes were protected from sulphydryl inhibition by NADH. -- Highly purified cytochrome b₅ reductase did not show any AH* or cytochrome c reductase activities. When purified cytochrome b₅ was added to the system, both AH* and cytochrome c reductase activities could however be demonstrated. DOC solubilization of microsomes, followed by DEAE cellulose chromatography, showed that AH* and cytochrome c reductases were resolved together and separated from a major ferricyanide reductase peak. -- When cytochrome b₅ was released from intact mitochondria and microsomes by trypsin treatment, the cytochrome c reductase was inactivated more effectively than the AH* reductase. -- The AH* and cytochrome c reductase showed similar lipid dependence. Both enzymes were inactivated by acetone extraction and reactivated by lipid micelles containing lecithin and oleic acid. Both enzymes showed similar sensitivity towards detergent inhibition. -- It is concluded that cytochrome b₅ reductase and cytochrome b₅ are components of the membrane bound AH* reductase. Certain dissimilarities observed between the AH* and cytochrome c reductases could be explained by an additional component of the AH* reductase not yet identified, which is not shared by the cytochrome c reductase. -- Possible functions of the AH* reductase have also been discussed. The enzyme may play a role in maintaining high levels of NAD in the cells of tissues that contain high concentrations of ascorbic acid, such as steroid producing tissues. The AH* reductase may also be an important protector of mitochondrial and microsomal membranes by reducing the semidehydroascorbate radical which is believed to be a powerful catalyst of lipid peroxidation. Thirdly, the enzyme probably has an important function in maintaining the ascorbate of the cell in a reduced state. Ascorbate may function in the cell as an enzyme cofactor.

Item Type: Thesis (Masters)
URI: http://research.library.mun.ca/id/eprint/7252
Item ID: 7252
Additional Information: Bibliography: leaves 77-84.
Department(s): Science, Faculty of > Biochemistry
Date: 1971
Date Type: Submission
Library of Congress Subject Heading: Enzymes; Liver extract

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